The overall aim of this proposal is to understand the role of the cAMP-specific phosphodiesterases (cAMP-PDEs) in hormone action. During the past funded period we have demonstrated that the cAMP- PDEs are regulated by FSH and TSH in a short term fashion via phosphorylation, and in a long term fashion via regulation of PDE gene expression. We have also demonstrated that multiple variants are derived from each of the PDE genes and that cAMP-PDEs are localized in specific subcellular structures. We now propose to continue these studies by investigating the role of these PDE variants in hormone action. The studies will be organized along three specific aims. In the first specific aim we will investigate the mechanism by which TSH causes a rapid activation of one of these PDE variants. The residues phosphorylated by TSH stimulation in the intact thyroid cell will be determined. Site directed mutagenesis will then be used to study the function of these phosphorylation sites in the enzyme activation. In the second specific aim, the role of the different PDE variants originating from the PDE4D gene will be investigated. A heterologous hormone responsive system expressing different cAMP- PDE variants will be used to study their impact on steroidogenesis and gene expression. After showing that cAMP-PDEs are targeted to different cellular compartments, the mechanism mediating this targeting will be investigated. In the third specific aim, the hypothesis that anchoring proteins target a PDE to different subcellular compartments will be tested by investigating the function of novel putative PDE-binding proteins that we have identified. These studies will further our understanding of the mechanism of TSH and FSH action in the endocrine target cell. They will also help define new targets for pharmacological intervention in endocrine disorders.